molecules

Review

The Effect of Vitamin Supplementation onSubclinical Atherosclerosis in Patients withoutManifest Cardiovascular Diseases: Never-endingHope or Underestimated Effect?

Ovidiu Mitu 1,2,* , Ioana Alexandra Cirneala 1,*, Andrada Ioana Lupsan 3, Mircea Iurciuc 4 ,Ivona Mitu 5 , Daniela Cristina Dimitriu 5 , Alexandru Dan Costache 2,† ,Antoniu Octavian Petris 1,2 and Irina Iuliana Costache 1,2

1 Department of Cardiology, Clinical Emergency Hospital “Sf. Spiridon”, 700111 Iasi, Romania2 1st Medical Department, University of Medicine and Pharmacy “Grigore T. Popa”, 700115 Iasi, Romania3 Department of Cardiology, University of Medicine, Pharmacy, Science and Technology,

540139 Targu Mures, Romania4 Department of Cardiology, University of Medicine and Pharmacy “Victor Babes”, 300041 Timisoara, Romania5 2nd Morpho-Functional Department, University of Medicine and Pharmacy “Grigore T. Popa”,

700115 Iasi, Romania* Correspondence: mituovidiu@yahoo.co.uk (O.M.); cirneala.ioana@gmail.com (I.A.C.);

Tel.: +40-745-279-714 (O.M.)† Medical Student, University of Medicine and Pharmacy “Grigore T. Popa”, 700115 Iasi, Romania.

Academic Editors: Raluca Maria Pop, Ada Popolo and Stefan Cristian VesaReceived: 25 March 2020; Accepted: 7 April 2020; Published: 9 April 2020

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Abstract: Micronutrients, especially vitamins, play an important role in the evolution of cardiovasculardiseases (CVD). It has been speculated that additional intake of vitamins may reduce the CVD burdenby acting on the inflammatory and oxidative response starting from early stages of atherosclerosis,when the vascular impairment might still be reversible or, at least, slowed down. The current reviewassesses the role of major vitamins on subclinical atherosclerosis process and the potential clinicalimplications in patients without CVD. We have comprehensively examined the literature data forthe major vitamins: A, B group, C, D, and E, respectively. Most data are based on vitamin E, Dand C supplementation, while vitamins A and B have been scarcely examined for the subclinicalatherosclerosis action. Though the fundamental premise was optimistic, the up-to-date trials withvitamin supplementation revealed divergent results on subclinical atherosclerosis improvement,both in healthy subjects and patients with CVD, while the long-term effect seems minimal. Thus,there are no conclusive data on the prevention and progression of atherosclerosis based on vitaminsupplementation. However, given their enormous potential, future trials are certainly needed for amore tailored CVD prevention focusing on early stages as subclinical atherosclerosis.

Keywords: subclinical atherosclerosis; cardiovascular diseases; vitamins; antioxidants; inflammation;primary prevention

1. Introduction

Multiple observational studies have shown an inverse relationship between increased levels ofmicronutrients and cardiovascular diseases (CVD). However, most interventional and prospectivetrials have not clearly confirmed the link between them, thus, CVD prevention guidelines do notyet recommend an additional intake of micronutrients. In contrast, new current research targetsthe link between micronutrients and early stages of atherosclerosis, respectively their influence on

Molecules 2020, 25, 1717; doi:10.3390/molecules25071717 www.mdpi.com/journal/molecules

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inflammatory markers given their antioxidant capacity, aiming the subclinical atherosclerosis as thepoint where the produced damage may be still reversible or at least slowed down with favorableresults over the targeted organs and systems.

Atherosclerosis, the leading cause of CVD, is an inflammatory pathology that begins whencholesterol-containing low-density lipoproteins (LDLc) accumulate in the vascular intima and activatethe endothelium. A complex immunological mechanism leads to the secretion of pro-inflammatorycytokines that contribute to the local inflammatory process and increase the atherosclerotic plaque.It is a process with a long asymptomatic evolution; the moment when symptoms occur denotesthe presence of advanced CVD. During the asymptomatic period of atherosclerotic progression, thesubclinical organ damage can occur. By applying measures of primary CVD prevention during thesubclinical atherosclerotic period, the incidence of CVD events can be decreased and slowed down,thus preventing the progression of pathological events [1].

Recent studies have focused on multiple mechanisms that appear to play key roles in theatherosclerotic inflammatory process, such as the oxidized LDLc that induces the recruitment ofmonocyte-macrophages to the endothelial wall, with highly atherogenic characteristics, the proliferationof smooth muscle cells induced by hyperglycemia, the oxidative stress and many others. Knowing theset off of these processes represents the finale purpose of these findings with the ultimate goal of stoppingor slowing them down in order to cure or delay the atherosclerotic process [2]. Certain diets maylead to the development of metabolic syndrome with a direct effect on the evolution of inflammatoryresponses of the vascular intima. Other studies hinted that micronutrient-full diets directly influencethe oxidative balance, the cellular growth and immune regulation [3]. Therefore, it appears thatmicronutrients, especially vitamins with antioxidant and anti-inflammatory properties, may play a keyrole in targeting the subclinical atherosclerosis by equilibrating the oxidation-antioxidation balanceand metabolism [4]. However, though the premises were consistent in many observational studies,most antioxidant and vitamin supplementation trials have failed to show an incremental CVD effect.Nonetheless, few studies have assessed the potential vitamin role in primary prevention and, especially,starting from subclinical modifications as early marker of CVD.

In the current literature review, we aimed to assess the influence of major vitamins on theprogression of subclinical atherosclerosis in patients without manifest CVD and their potential effecton reducing the CVD burden starting from subclinical disease. For this purpose, we have introducedcertain keywords (subclinical atherosclerosis, each vitamin separately, cardiovascular diseases, prevention)in the major databases (PubMed, ScienceDirect, Web of Science). The obtained papers were furtherreviewed in order to exclude subject groups with manifest or atherosclerotic CVD while the remainingarticles have been extensively analyzed.

2. Vitamins and Subclinical Atherosclerosis

2.1. Vitamin E

Vitamin E is an essential micronutrient that includes both tocopherols (TP) and tocotrienols (T3),which comprise antioxidants that are meant to modulate lipid peroxidation. It is found in plants, seedsand their derivatives. The most active isomer of this element is α-tocopherol, a very liposoluble phenol,with an important antioxidant action [5], making it particularly important in the human metabolism asit appears to be found in fat deposits, lipoproteins, and tissues with lipid-rich elements [6].

This vitamin appears to be involved in several stages of inflammation and immune regulation,modulating cell functions and gene expression [7]. It has protective effects against CVD, metabolicdisorders and other diseases where oxidative stress represents a risk factor, by cancelling the oxidationproduced by free radicals on cells, nervous tissues or membranes [8]. Due to the presence of tocotrienols,it has the effect of lowering cholesterol beyond the antioxidant mechanism [7–9].

Chronic and continuous inflammation is involved in the evolution of atherosclerosis. The oxidativestress produced inside the cell is a key mechanism in the oxidative damage of biomolecules. Thus, it is

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understandable why a clear link between vitamin E and subclinical atherosclerosis is under research,being widely suggested that it can provide a useful prognosis in CVD and metabolic disorders as wellas a therapeutic agent in various pathologies. Many animal studies showed the beneficial effects ofTP and T3 on vascular lesions of atherosclerosis, but in clinical trials the results were variable, mostof them negative [10,11]. Though the reasons for these results were unclear, it was supposed thatresearch animals can be better controlled and more rigorously measured than humans in trials [10].The advanced stage of atherosclerosis or CVD was another hypothesis for the high doses of vitamin Ethat were required for showing an improvement in the studied pathology [11,12].

In the prevention field, though, vitamin E showed a better response. In the early stage ofatherosclerosis, the required quantity was lower and, even though the patients could not be drasticallycontrolled, the correlation between serum vitamin E and the decrease of LDLc and total cholesterolwere easily observed [13]. Serum lipid concentration is a paramount factor in the development ofatherosclerosis, dyslipidemia being strongly related to CVD.

Vitamin E, due to its antioxidant properties, protects hepatic cells against oxidative stress [14].A recent study aimed to correlate circulating α- and γ-TP levels to adiposity-related characteristics byusing high performance liquid chromatography and magnetic resonance imaging to quantify the liverfat and adipose tissue. Though the serum vitamin E level depends on the variable absorption in thegastro-intestinal tract [15], the vitamin status can be influenced by the liver metabolization and theoxidative stress. There was a significant association between the metabolic alterations and the serumconcentration of the studied micronutrient [16,17].

Another study assessed the vitamin E supplementation for a 3-year period in two groups –smoking men, otherwise healthy, and post-menopausal women. The preventive effect was moreobvious in the first group. This fact may be generalized to all male subjects, however, the lack ofeffect on women is not definitely, further studies being necessary in order to consider either this resultmay be due to statistical error or because this group has better protection against atheroscleroticdevelopment [18].

Trials revealed that patients with subclinical atherosclerosis (with risk factors but no CVD) thatwere treated with vitamin E showed significantly improvement of peripheral-artery disease and adecrease incidence of angina pectoris. No trial showed beneficial results on the carotid intima-mediathickness (cIMT) progression in secondary prevention CVD patients [19].

Vitamin E has been one of the most studied antioxidants that may be involved in CVD prevention.Many observational and cohort studies suggested an inverse association between high dietary intakeof vitamin E supplementation and CV events [20–22]. Though the postulated benefit was rathersmall to medium, there was a promising premise for implying vitamin E into CVD prevention. Theantiatherogenic properties of vitamin E were suggested in its ability to decrease LDLc, diminishoxidative stress, reduce inflammation and inhibit vascular smooth muscle cell proliferation andexpression of adhesion molecules [21].

Nonetheless, the interventional CVD trials of vitamin E supplementation presented divergentand modest positive results and did not fulfill the initial expectations [20,22]. As well, the associationbetween CVD and serum or plasma TP level was rather inconsistent. Several hypotheses have beenpostulated for the lack of efficacity in interventional trials [22,23]. First, self-reported nutritionalquestionnaires are highly subjective and usually people tend to over-report their intake of fruits andvegetables or under-report other sources of vitamin E such as oil consumption. Secondly, it is difficultto quantify precisely the beneficial amount of vitamin E as there is a high bioavailability of TP andT3 in a mixture of foods that contribute to the daily nutrient intake and the interactions with otherfood ingredients could represent another issue. Further on, in nutritional interventional trials a greatnumber of different CVD-associated parameters may appear and overrule the potential beneficial effectof vitamin E supplementation on CVD outcomes. On the other hand, high doses of vitamin E mayexhibit prooxidant effect by enabling production of α-tocopheroxyl radical [21].

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Most interventional trials failed when using vitamin E in secondary prevention but in primaryprevention the effects seem to be encouraging. Thus, some interventional studies intended to assess thepossible role of vitamin E supplementation on subclinical atherosclerosis as early marker of dysfunctionas primary prevention CVD surrogate [18,24–26]. Though subclinical CVD modifications cannot beextrapolated to CVD outcomes, the results are encouraging and have been summarized in Table 1.

Table 1. Effects of vitamin E supplementation on subclinical atherosclerosis in patients withoutmanifest CVD.

Trial Year Follow-up Dose Population Age Results

Rasoolet al. [24] 2008 2 months

112, 224, 448IU vitamin E,

daily

36 healthysubjects 23.9 ± 0.39 years

Improvement of pulsewave velocity (PWV)at doses of 224 and

448 IU/day

Hodis et al.(VEAPS) [25] 2002 3 years

400 IUvitamin E,

daily

353 healthysubjects with

high LDLc

56.2 years (range40–82 years)

No effect on cIMTprogression

Skyrme-Joneset al. [26] 2000 3 months

1000 IUvitamin E,

daily

41 type 1diabeticsubjects

23 ± 6 years

Improvement ofendothelial

vasodilatationfunction

Salonen et al.(ASAP) [18] 2000 3 years

272 IUvitamin E,

daily

115 healthysubjects range 45–69 years Improvement of cIMT

2.2. Vitamin D

Vitamin D is a group of lipophilic prohormones synthesized in the skin with the aid of ultraviolet-Bradiation exposure and activated into the liver and kidneys by sequential hydroxylation or obtained bydietary means and absorbed by the lymphatic system from the proximal small intestine. It facilitates theabsorption of calcium in the bowels and the deficiency of this vitamin can be associated with significantpathological changes in CV structure, mediating the link between parathyroid, kidneys and bones.Vitamin D therapeutic roles include improvements in endothelial function via anti-inflammatoryactions [27].

Low vitamin D is related to a high CV risk, affecting proatherogenic serum cholesterol loadingcapacity, adipokine profile and subclinical atherosclerosis. Such results are achieved since thesevasculotropic hormones present a wide expression of receptors in many body cells and organs [28].

The anti-atherosclerotic effect of vitamin D is explained by the positive impact on circulatinglipoproteins functions, which play a key role in atherogenesis, rather than their serum concentration.The modulating effect on serum lipoprotein functions is closely linked to macrophage cholesterolhomeostasis. Vitamin D also reduces the serum cytokines and adipokines, well known proatherogenicmediators, having a beneficial effect on adipokine profile, lipoprotein functions and vascularparameters [28].

By these mechanisms, vitamin D ameliorates vascular health and lowers the CV risk. Differentstudies have revealed that this hormone protects against atheromatous disease in patients with chronickidney disease. In dialyzed patients, a connection between body mass index (BMI) and vitamin D wasobserved: lower odds of atheromatous progression were found in patients with low to moderate BMIbut with higher levels of serum vitamin D [28].

Low serum concentration of 25-hydroxyvitamin D [25(OH)D] increased atherosclerotic and CVrisk, being associated with dyslipidemia, high levels of serum LDLc and low levels of high-densitylipoproteins (HDLc) in pre-menopausal healthy women [29,30]. The supplementation amelioratedserum lipoprotein functions, adipokine profile and subclinical atherosclerosis. As markers ofatherosclerosis, standard measurement techniques were used, such as flow-mediated dilatation(FMD), pulse wave velocity (PWV) and augmentation index (AIx). The deficiency was defined asserum levels of 25(OH)D under 20 ng/mL. After the exclusion of patients with history of CVD and risk

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factors, or patients that received drugs that affect the endothelial functions such as statins, nitrates orangiotensin-converting enzyme inhibitors, the study concluded that vitamin D supplementation has akey role in lipoprotein functions in real life situation [31].

There are gender-differences in the action of vitamin D. A South Korean study that studied theserum levels of 25(OH)D on 404 men and 650 women revealed consistent discrepancies on metaboliccomponents such as blood pressure, lipid profiles or glycemic index as well as on arterial changes suchas brachial-ankle PWV (baPWV) or cIMT. While men did not present a particular correlation, womenshowed a connection between dietary vitamin D and triacylglycerol levels while both sexes showed adirect relationship between vitamin D and HDLc, but this study did not reveal a beneficial improvementon subclinical atherosclerosis despite vitamin D direct actions in the cholesterol metabolism [32].

Few studies covered the process of atherosclerosis in young people, given the idea thatatherogenesis begins early in childhood. Risk factors, such as obesity, accelerate the atheroscleroticprocess, cIMT being a subclinical indicator of atherosclerosis, especially among overweightindividuals [33]. Vitamin D concentration is associated with parathyroid hormone (PTH) thatcontrols bone metabolism and has also receptors on arteries. Thus, high serum concentrations leadto hypertension, cardiac hypertrophy and endothelial dysfunction [34]. Moreover, vitamin D haseffects on vascular smooth cell muscle, increasing the risk of atherosclerosis, while low concentrationcan lead to exacerbation of other CVD risk factors, such as arterial hypertension, obesity or diabetes.Even though there is limited evidence, a link between low 25(OH)D concentrations in childhood andincreased cIMT in adulthood has been described. The study concluded that there may be a directcorrelation between vitamin D insufficiency and subclinical atherosclerosis in overweight adolescents,but further investigations are needed in order to elucidate the possibility that PTH may contributeindependently of 25(OH)D in the atherosclerotic process [34].

For quantifying the evidence that indicates the association between vitamin D and increased riskof CVD, the incidence of hospitalization for heart failure in patients with low concentration of 25(OH)Dhas been studied. Compared to normal levels of vitamin D, low serum concentration of 25(OH)D wasassociated with higher risk of heart failure hospital admission and with more days of hospitalization.It can be speculated that vitamin D cannot undo the existing lesions but may prevent further CVDevolution and decrease symptoms [35].

Though all these mechanisms, the supplementary intake of vitamin D was assumed to have aprotective effect on CVD development. A large meta-analysis comprising more than 26.000 individualsfrom eight prospective cohort studies revealed a consistent association between 25(OH)D level andCV mortality in subjects both with and without a CVD history at baseline [36]. However, in primaryCVD prevention, the vitamin D implications seem to diminish the clinical relevance. As a vitaminthat is highly available, many studies tried to assess CVD outcomes or subclinical CVD modificationswith an increase intake of vitamin D. The inconsistences of vitamin D trials could be attributable toheterogeneity in dosage or baseline concentrations, different non-accountable CVD biases or otherconcomitant treatment, study duration or design, or population type [21,37]. There is no standardconsensus for vitamin D administration, especially for the CVD prevention.

The up-to-date study results of vitamin D supplementation on subclinical atherosclerosis arerather controversial (Table 2). There is no certain proof that vitamin D can induce regression of alreadypre-existing lesions and the effects of supplementation on different pathologies are not clarified but ithas been demonstrated that it may ameliorate vascular health in subjects with low CV risk. Nonetheless,the genetic status and lifestyle factors may overrule the contribution of vitamin D regarding the risk ofdeveloping subclinical atherosclerotic disease [38].

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Table 2. Effects of vitamin D supplementation on subclinical atherosclerosis in patients withoutmanifest CVD.

Trial Year Follow-up Dose Population Age Results

Borgi et al. [39] 2017 8 weeks 50.000 IU D284

overweight/obeseadults

37 ± 12.3 years No improvement onendothelial function

Kumar et al. [40] 2017 16 weeks 2 doses of300.000 IU D3

117 subjectswith chronic

kidney disease43.17 ± 11.79 years Improvement on

FMD and PWV

Raed et al. [41] 2017 16 weeks 600, 2000, 4000IU/day D3

70 overweightpatients

26.2 ± 9.8, 24.4 ± 8.7,25.5 ± 9.0 years

Improvement onPWV

Bressendorff et al. [42] 2016 16 weeks 3000 IU D3 40 healthysubjects 41.0 ± 9.05 years No effect on PWV

Farouhi et al. [43] 2016 16 weeks 100.000 IU D2 /100.000 IU D3

160 type 2diabetic subjects

53.5 ± 8.7 years (D2);52.5 ± 8.2 years (D3)

Improvement onPWV

Zaleski et al. [44] 2015 6 months 400 IU/day vs.4000 IU/day D3

40prehypertensive

subjects

34.8 ± 12.8 years (lowdose); 40.4 ± 7.5 years

(high dose)No effect on PWV

Martins et al. [45] 2014 12 weeks 100.000 IU D3130

overweight/obesesubjects

range 18–70 years No effect onaugmentation index

Breslavsky et al. [46] 2013 1 year 1000 IU D3 32 type 2diabetic subjects 69 ± 9 years Improved

augmentation index

You et al. [47] 2013 3 months 5000 IU D3 12 type 2diabetic subjects 65 ± 8 years No effect on FMD

Harris et al. [48] 2011 4 months 60.000 IU D3 45 overweightpatients 29 ± 2 years Improved FMD

Sugden et al. [49] 2008 8 weeks 100.000 IU singledose D2

34 type 2diabetic patients 64.9 ± 10.3 years Improved FMD

2.3. Vitamin C

Vitamin C, also known as ascorbic acid, in an essential water-soluble nutrient with antioxidantproperties. The mechanism by which cellular DNA, lipids and proteins are protected from theoxidative stress and the damage of radical oxygen species (ROS) is still unknown. It is certain thatthe anti-inflammatory properties of vitamin C explain the preventive effects of the diets rich in thisnutrient in CVD or cancer [50]. Ascorbic acid has a key role in many physiological processes such asantioxidation, antiinflammation and oxidative stress. The vitamin C property of donating electrons isclosely linked to all of its known functions [50].

Low plasma concentrations of vitamin C inevitably lead to scurvy. Signs and symptoms ofthis disease are related to the well-known enzymatic actions of ascorbate, such as the difficulty ofcollagen binding that results in loss of teeth and tissue damage, as a reflection of connective tissuedestruction [51]. This is due to the ascorbic acid properties that participate in the synthesis of cholesterol,peptide hormones, amino acids, catecholamines and carnitine. Though these enzyme actions have notbeen unequivocally demonstrated in vivo, there must be some biochemical pathways that specificallyrequire vitamin C, otherwise scurvy could be cured without ascorbate [52].

The enzymes used in the hydroxylation of lysine and proline (residues of collagen) – reactionwhich is a mandatory process for the evolving of the collagen triple helix physiological evolution – areclosely linked with ascorbic acid [53,54].

Many diseases including atherosclerotic CVD or cancer may appear as a cause of the oxidativedamage of the cells [50,55]. The genesis of atherosclerosis depends on multiple factors that are presentalongside with other CV comorbidities such as diabetes mellitus, metabolic syndrome, dyslipidemiaand arterial hypertension [56,57].

Different studies revealed that the use of antioxidant vitamin supplementation might reduce theCVD risk. Initial results showed the positive connection between the vegetable and fruit intake andimprovement in CV outcomes. Further data demonstrated that the patients consuming such foodhad a lower risk of coronary heart disease (CHD) unlike people who were on the lowest quintile offruits and vegetables intake [58]. The next step was pointing out which nutrient has the highest effecton CV physiology. Vitamin C was thoroughly studied because of its antioxidant capabilities and

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the direct effect on oxidized LDLc, which is quickly incorporated into the atherosclerotic plaque byscavenger receptors [59,60]. These findings made the LDLc oxidation as target of prevention, withvitamin C showing a potential role in CVD prevention. As well, it presents the capacity of reducingthe monocytes endothelial adhesion, mechanism that is considered to be one of the early signs in thedevelopment of atherosclerosis. This hypothesis has been explored in cigarette smoking individuals,revealing that restoring the plasma vitamin C concentration in people that smoked one to two packs ofcigarettes per day decreased the ability of monocytes to adhere on the endothelial wall as compared tothe values found in non-smokers [61].

Additionally, ascorbic acid showed an increased action on nitric oxide, improving the latterproduction and reducing blood pressure. A study conducted on younger and older healthy adultsdemonstrated a significant reduction of arterial stiffness by measuring the PWV but there was acontroversial lowering of systolic and diastolic blood pressure in older subjects and an increaseof diastolic blood pressure in the younger patients when compared a regime of acute vitamin Cadministration to a chronic supplementation. The acute vitamin C administration was composed ofa bolus of 0.06 g/kg fat-free mass (FFM) dissolved in saline and infused for 20 min, followed by a“drip infusion” of 0.02 g/kg FFM over 60 min while the oral supplementation was administrated for 30consecutive days, 500 mg/day. Another novel aspect was represented by the spectacular increase ofascorbic acid concentration in younger subjects with initial lower concentrations as compared to theolder subjects, but a clear explanation has not been defined yet. A plausible hypothesis might be thatthe renal function is reduced with age, resulting in slower kidney excretion of the nutrient excess inolder subjects. However, further investigation on this matter is required [62].

Moreover, vitamin C appears to prevent the apoptosis of vascular smooth muscle cells withan important role in keeping the atherosclerotic plaque more stable. Oxidized LDLc is toxic for themacrophages, smooth muscle cells and endothelium, with a tremendous role in the initiation anddevelopment of atherosclerosis, being also localized within the atherosclerotic lesions [63]. Anotherstudy investigated the cytoprotective effects of ascorbic acid by inducing cell apoptosis of humansmooth muscle cells and then observing the attenuation effect of pre-administration of vitamin C,providing protection against cytotoxicity of LDLc and lipid hydroperoxides [64].

The recommended intake levels of vitamin C of 75 mg for women and 90 mg for men, per day,may not be enough given the quantity of dietary ascorbic acid needed to have a therapeutic effect inCVD. Most studies used supplementation doses of 500–1000 mg vitamin C per day in order to achieverelevant results [65,66].

To evaluate the oxidative stress on endothelium, another research aimed for the effects of vitaminC on blood pressure in dipper and non-dipper hypertensive patients. Under controlled conditions,vitamin C was administered before the intraarterial infusing with acetylcholine, saline and sodiumnitroprusside at increasing doses. This trial showed that ascorbic acid protects the vascular wall fromfree radical lipid peroxidation and from vasodilatation induced by acetylcholine [67].

Vitamin C acts as an antioxidant and as an enzyme cofactor, with primary biological effects.Oxidized LDLc is highly atherogenic and induces the recruitment of monocyte-macrophages to theendothelial wall, which is a key mechanism in the subclinical atherosclerotic onset. Ascorbic acid effectmay start in early stages of vascular dysfunction and, as a result, of atherosclerosis, though vitamin Cseems to improve the inflammatory status or blood pressure [68,69].

On the other hand, long time supplementation with vitamin C had no effect on major CV eventsin specific clinical trials, while even one study remarked a higher all-cause mortality in the antioxidantgroup but on a population with present CVD [66,70]. The initial hypothesis was sustained by somecohort studies that generally showed an inverse correlation between vitamin C level and populationwith manifest atherosclerotic CVD or diabetes [66,71]. The lack of data consistency should be regardedfrom different aspects: apparent different effect of dietary vitamin C from supplementation, ratherlimited follow-up, reliance on self-reported nutritional questionnaires, as well as the presence ofmultiple human antioxidant mechanisms that have not been fully understood. The low vitamin status

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may allow a faster progression of the disease so the expected effects in subjects with normal plasmaconcentrations of vitamin C seem minimal [72]. However, some data show a direct link betweenvitamin C and endothelial function as it may reduce the arterial stiffness through antioxidant andanti-inflammatory effects leading to potential stabilization of the atherosclerotic plaque [21,71]. Basedon such premises, there are currently few studies that examined subclinical modifications in personswithout CVD, but the results for vitamin C look promising and plead for further research (Table 3).

Table 3. Effects of vitamin C supplementation on asymptomatic CVD subjects andsubclinical atherosclerosis.

Trial Year Follow-up Dose Population Age Results

Hildreth et al. [73] 2014 6 months

0.06 g/kg offat free

mass–max7.5 g vitamin

C,intravenoussingle dose

97 healthywomen 22–70 years

Improvement ofarterial stiffness

(measured by carotidartery compliance)

Mullan et al. [74] 2002 4 weeks500 mgvitamin

C/day, orally

30 type 2diabeticsubjects

61.0 ± 6.5years

Improvement ofarterial stiffness

(augmentation index;time to wave

reflection)

Wilkinson et al. [75] 2001 6 h2000 mg

vitamin C,orally

8 healthysubjects

29 (20–42)years

Improvement ofaugmentation index,

but not on PWV

2.4. Vitamin B

B vitamins are a complex of eight water-soluble compounds with chemically distinct structureand essential role in the cell metabolism as they act as coenzymes in a variety of anabolic and catabolicenzymatic reactions [76]. They often coexist in the same type of food, but individual B supplementscan be used separately, as each one presents specific properties, being either a cofactor for essentialmetabolic processes or a precursor in the same area [77]. The following paragraphs aim to assess thepotential role of each vitamin B subgroup on the atherosclerotic process.

2.4.1. Vitamin B1, or Thiamine (Aneurin)

This is a coenzyme with an active role in processing sugar, fats and amino acids. This micronutrienttakes four forms in the human body and is essential in many oxidation-reduction reactions such as theKrebs cycle (for adenosine triphosphate production) or the glucose metabolism. It can be obtainedfrom many types of food, such as cereals, red meat, seeds, nuts, yeast, but high temperature and pHdenaturate thiamine, as well as food processing, losing all its beneficial properties [78].

Thiamine loss is closely connected with creatinine clearance since the serum excess that is notbound with protein is eliminated through the distal nephrons. This is the explanation why diureticsare the main cause of thiamine deficiency in patients with CVD. Moreover, the levels of serum thiaminepresented a negative correlation with triglycerides and LDLc [79].

In the development of atherosclerotic plaque, an essential role is represented by the proliferationof arterial smooth muscle cells through glucose and insulin mediated mechanisms. Thiamine, asan essential element in glucose metabolism, has a protective role against the atherogenic process,counteracting the effect of high glucose doses that have a tremendous damaging effect over the vascularwall by inducing chronic inflammation through multiple pathways, such as lipid peroxidation, injuryor infection that tend to increase the risk of dyslipidemia [80].

An improvement in cardiac and hemodynamic functions as well as a decrease in vascular resistancehave been detected after the administration of intravenous thiamine. In patients with hyperglycemia, the

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endothelium-dependent vasodilatation improved, being recommended the administration of thiamineas routine for slowing down atherogenesis and for the improvement of endothelial functions [81].

A recent trial assessed the endothelium-dependent vasodilatation in three categories of patients:healthy, with impaired glucose tolerance, and with non-insulin-dependent diabetes using duplexultrasound to measure brachial artery vasoactivity. The study concluded that the effects of thiamine arenot based on the glucose-lowering mechanism, given that there was no change under normoglycemia.However, in hyperglycemic patients prone to accelerated atherosclerosis, the effects of vitamin B1 wereoutstanding by improving the endothelial function and lowering the development or the progressionof subclinical atherosclerotic disease [81].

Other studies revealed that supplementation with vitamin B1 has dramatically reduced the vascularinflammation, with negative correlations between high levels of thiamine and LDLc, respectively,triglycerides. Therefore, it may be considered that chronic administration of vitamin B1 can delay theatherosclerotic process [82].

Vitamin B1 can be used both as a marker and as a therapeutic element, with presumed role in thedevelopment and also in the prognosis of atherosclerotic vascular pathology. Routine administrationcould improve vascular function in hyperglycemic persons that represent a target group in many trialsassessing subclinical atherosclerosis [83].

2.4.2. Vitamin B2, or Riboflavin

Riboflavin is a water-soluble vitamin, essential for human health, that is closely connected withenergy production by acting as a cofactor through its two forms: flavin adenine mononucleotide (FMN)and flavin adenine dinucleotide (FAD). It is found in a wide variety of foods, mostly meat and dairy,most of the time in its cofactor forms, attached to proteins. Deficiency of vitamin B2 is more prevalentin under-developed countries and often occurs along with other nutrient deficiencies [84].

The antioxidant effects of riboflavin have not been widely studied. However, research showed thecritical action of vitamin B2 in its FAD coenzyme form in the glutathione redox cycle. An increase ofoxidized glutathione can lead to cellular damage and may occur during riboflavin deficiency meaningthat an increase in lipid peroxidation can occur, with direct effects in the atherogenic pathways [84].

Many animal studies have shown improvement in subjects with riboflavin deficiency, but in theonly human trial that was conducted, there was no significant difference between healthy subjectsand riboflavin-deficient subjects [85]. Still, riboflavin status affects the production of lipid peroxides,which means that oxidative stress is closely connected to vitamin B2 [86]. These findings prove thatriboflavin has a key role in human body defense system as an antioxidant and the low intake can affectthe atherosclerotic process by misbalancing the oxidant-antioxidant ratio [87].

2.4.3. Vitamin B3, or Niacin or Nicotinic Acid (NA)

Niacin is a water-soluble vitamin with essential role in cellular energy and metabolism. It isfound in a large variety of foods such as meat, grains and dairy. Niacin deficiency is rare in developedcountries and can lead to pellagra, a human disease characterized by symptoms such as high sensitivityto sunlight, dermatitis, diarrhea, depression and dementia [88].

NA has an essential role in metabolism as it is the precursor of nicotinamide adenosine dinucleotide(NAD+) and nicotinamide adenine dinucleotide phosphate (NADP+) that have key roles in glycolysis,tricarboxylic acid cycle, the respiratory chain and other metabolic processes that require electrontransport. They also have a major impact in cellular processes such as DNA repair and genomicstructure [88].

Niacin deficiency is involved directly in the oxidative stress by altering the redox balance of thecell, explaining thus the decreased levels of antioxidant enzymes and by interfering in the regenerationof glutathione, with a direct effect on deactivating peroxides as a response to stress [89].

The effect on subclinical atherosclerosis has been intensively studied since the revealing ofthe niacin ability to increase plasma HDLc and to decrease the levels of proatherogenic lipids and

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lipoproteins, thus intervening in the atherogenic process. HDLc is an independent risk factor in CVD,with predictive capacity in the atherosclerotic vascular disease, with lower levels found especially inhealthy women and less in men [90]. NA also prevents endothelial dysfunction by improving theresponse to proinflammatory and prothrombotic factors of the vascular wall and by its effect over thesubstances released from the endothelial cells that inhibit vasorelaxation [91]. Niacin hypolipidic effectalso reduces blood thickness and antiplatelet aggregation, explaining the direct effect on subclinicalatherosclerosis [92].

Many studies have focused on niacin effect on atherosclerotic plaque, most of them usingassociations with statins or other pharmaco-active substances in CVD area, all of them with promisingresults [93]. However, only one trial used niacin alone versus placebo. The study was conducted over52 weeks and on 50 patients with metabolic syndrome but no CVD, randomized to either placeboor 1 g extended-release niacin. After this time period, by cIMT assessment, the endothelial functionincreased by 22% in the niacin receiving group, while no change was seen in the placebo group. Thus,therapy with extended-release niacin may improve the metabolic parameters, by increasing HDLc andreducing triglycerides, with effects on atherosclerotic plaque reduction [94].

2.4.4. Vitamin B5, or Pantothenic Acid (PA)

Pantothenic acid is a precursor for coenzyme A (CoA), essential for biochemical reactions in humanbody such as Krebs cycle, fatty acid metabolism or β-oxidation [95]. The role of PA in atherogenesisis not fully elucidated, but it may have a key effect on reduction of oxidative stress by increasingglutathione synthesis [96].

C reactive protein (CRP), a circulating inflammatory marker, has a controversial role in theatherosclerotic disease, but it can transform oxidized LDLc into foam cells and can be a stablebiomarker for low-grade inflammation, especially found in subclinical atherosclerosis. Several studieshave shown a cross-link between the levels of PA and CRP as well as inverse correlation with LDLcand triglycerides levels, but the mechanisms are still unknown [97,98].

Pantethine is physiologically synthesized in the human body from PA and is safe and effectivein low and moderate CVD risk, being used as nutritional supplement since 1992, as it showed asignificant lowering of LDLc and total cholesterol serum concentrations [99]. Data revealed that a16-week treatment with pantethine reduced LDLc and total cholesterol as compared to placebo, with asignificant decrease of the lipid profile in women. Women also had a better decrease of non-HDLclevel compared to men who had better results on LDLc fraction [100].

High concentrations of plasma lipids play an essential role in atherogenesis and progressionof atherosclerosis. Vitamin B5, as a precursor of CoA, favorably affects triglycerides synthesis andlipoprotein metabolism, contributing to the onset and development of subclinical atherosclerosis.

2.4.5. Vitamin B6

This is a water-soluble vitamin that can be found in various foods such as meat, whole grains,vegetables, fruits and nuts. It has six isoforms, pyridoxal 5′-phosphate (PLP) being the most active.PLP acts as coenzyme for various reactions such as amino acid and carbohydrate metabolisms. VitaminB6 may have proved a preventive role in CVD, stroke and thrombosis [101–103].

Still, trials must prove that low serum vitamin B6 is an independent risk factor for CVD. Lowserum PLP appeared to be connected with the imbalance of lipid metabolism. With a direct role inthe metabolism of homocysteine, which is involved in atherothrombotic vascular disease by causingdirect endothelial damage, increasing oxidative stress, negatively altering the endothelial function andthrombogenesis, PLP might have a multifactorial influence on CVD [104].

Furthermore, PLP is also associated with circulating CRP and other oxidative stress markers thatare known to reduce superoxide radical effects in endothelial cells. Also, low vitamin B6 concentrationsdecrease the glutathione, alter the antioxidant defense system and lead to inflammation, a crucialmechanism in subclinical atherosclerosis [105,106].

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2.4.6. Vitamin B7, or Biotin

Biotin is a vitamin found naturally in meat, eggs, cereals and vegetables. It is a cofactor for avariety of carboxylation reactions and is involved in fatty acids metabolism and gluconeogenesis [107].Biotin deficiency is extremely rare, and even though it might influence the prevention of atherogenesisor atherosclerosis, there are no current studies to prove that yet [108].

2.4.7. Vitamin B9, or Folic Acid

Folic acid presents a beneficial role after acute or chronic administration in various CV predictivemarkers such as blood pressure, arterial stiffness, endothelial function and pro-thrombotic activity bylowering homocysteine levels and influencing the endothelial nitric oxide synthase (eNOS) [109].

Folic acid also has independent effects on atherogenesis. Chronic administration enhanced theendothelial function, but the homocysteine concentration did not change, these effects being mediatedby folic acid and eNOS [110].

Research data support the premise that vitamin B9 may be an effective therapeutic option to reducethe inflammatory response in subclinical atherosclerosis by decreasing the plasma concentration ofhomocysteine or by the direct interaction with eNOS to reduce production of reactive oxygen species.

Trials on cigarette smokers, otherwise healthy patients, showed that supplementation with folicacid on a 4-week trial reduced significantly the concentration of homocysteine and fibrinogen ascompared to placebo [111].

Another trial used 1-year folic acid supplementation in order to explain the correlation betweenvitamin B9, hyperhomocysteinemia and the proinflammatory status in high CVD risk. 530 menand postmenopausal women with hyperhomocysteinemia at the beginning of the study have beenrandomized. The effect on inflammatory markers was not significant, revealing that it might be anothermechanism for vitamin B9 protective effect [109,112].

2.4.8. Vitamin B12, or Cobalamin

Cobalamin is a water-soluble vitamin used in cell metabolism and is naturally acquired throughdiet, especially from animal products, being essential for normal functioning of all human tissues. Fora good absorption, the stomach, pancreas and ileum must be physiologically intact, otherwise vitaminB12 deficiency may occur [113].

Homocysteine and methylmalonic acid are metabolites of cobalamin, with key roles in atherogenicprocesses. Low levels of serum vitamin B12 have direct effects on subclinical atherosclerosis bymediating reactions such as the extraction of energy from fat and protein in the mitochondria or theconversion of homocysteine to methionine. These mechanisms reveal how multiple organ-systemscan be affected by vitamin B12 deficiency. The clinical manifestation is linked to the severity oforgan-system damage but is usually asymptomatic [114].

Hyperhomocysteinemia associated to nutritional deficiency is mild but linked with a high risk ofatherogenesis induced by endoplasmic reticulum stress, proinflammatory response and oxidative stress.A study on 158 healthy siblings of patients with premature atherothrombotic disease revealed thatlowering the homocysteinemia by cobalamin administration showed a decreased risk of atheroscleroticevents as compared to placebo group [115].

By comparing genders, high concentrations of homocysteine were found in males that possesseda higher risk of atherothrombotic process development. However, lowering serum homocysteineconcentration was equal in both males and females, with good appliance for future therapeuticalapplications of vitamin B12 [116].

Even though signs and symptoms of vitamin B12 deficiency are not specific enough for a fastdiagnosis, prompt vitamin administration is crucial before irreversible lesions install. Accuratediagnosis remains controversial and the screening is not reliable. Nonetheless, since the treatment is

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easy and safe, vitamin supplementation should be established at least in persons with risk factors ofdeveloping atherothrombotic vascular disease [113,117].

Overall, to the best of our present knowledge, there is no study that has established a specific Bvitamin supplementation effect on subclinical atherosclerosis evolution in patients with no CVD atbaseline. However, in most clinical trials, there was given a “cocktail” of vitamin B subtypes, most ofthem including B6, B9, B12. Most interventional studies regarded patients with advanced renal diseaseas folic acid and B12 may have beneficial effects on renal disease.

The meta-analysis results showed that vitamin B supplementation had no effect on loweringCVD events though, in some subgroup of patients, a higher intake of vitamin B seemed to offer abenefit in primary CVD prevention but not in secondary prevention [118]. The inconclusive results ofobservational and interventional trials with vitamin B might be explained though the different typesand doses of supplements, the non-standardized patient evaluation, multiple CVD comorbidities anddivergent types of population. However, hopeful data came from the “B-Vitamin AtherosclerosisInterventional Trial” (BVAIT) that aimed to demonstrate whether homocysteine is a marker or thecause of atherosclerotic vascular pathology given that high levels of this amino acid are associatedwith high CVD risk. By measuring cIMT and the aortic and coronary artery calcium score, high dosesof vitamin B supplementation (composed of folic acid 5 mg + vitamin B12 0.4 mg + vitamin B6 50 mg)reduced the progression of subclinical atherosclerosis in patients with no CVD [119].

2.5. Vitamin A

Vitamin A (all-trans-retinol) is a provitamin found in vegetables that exists in different metabolicstates: retinal (retinaldehyde), oxidized forms and conjugations of retinol and retinoic acid. The termretinoid was introduced in 1970 and gathers under its name all chemical compounds that structurallyresemble all-trans-retinol, with all its metabolites, both natural and synthetic [120,121].

For long time, vitamin A actions used to be focused on proliferative disorders, mainly aroundcancer and dermatologic diseases. However, new findings have proved that vitamin A possesses certaininfluence on the CV system and metabolic pathologies, being involved in angiogenesis, oxidativebalance and cellular growth [122].

Vitamin A is not a water-soluble micronutrient, being itself a lipid. This explains why it is mainlyfound bound to specific vitamin A-binding proteins or within the cells in lipid droplets, proteins thatare highly involved in the pathogenesis of obesity and diabetes [122]. The processing of vitamin A inthe intestine after dietary intake is similar with triglycerides and cholesterol, but once absorbed, themetabolism, as well as its storage, is unique. Vitamin A is essential for mediating various physiologicalprocesses in the human body, some of them related to cholesterol and triglycerides metabolism,contributing to pathogenesis of metabolic diseases when dysregulated [123].

There is a connection between retinoids, the arterial responses to injury and atherogenesis.Animal studies proved an impressive decrease of atheromatous plaque after four days of vitaminA administration as well as a reduction of the neointimal injury by modulating smooth muscle cellmigration and proliferation with essential role in atherogenesis [124]. Retinoids limit the vascularsmooth muscle cell proliferation and regulate apoptosis, while lesions appear when there is animbalance between smooth muscle cell proliferation apoptosis and cell death [125]. Retinoids also havea direct role in the coagulation system by promoting fibrinolysis and decreasing the pro-thromboticeffects of the vascular endothelium [126]. Furthermore, vitamin A regulates favorably the inflammatorymechanisms by decreasing endothelial adhesion molecule expression with a direct role in all stages ofatherogenesis [127].

In observational and interventional cohort studies, vitamin A supplementation was not associatedwith incremental effect on CVD events [128]. Nonetheless, there is data that suggest that the protectiveeffects of vegetables and fruits against CVD may be due to increase lutein consumption. It presentsantioxidant properties and prevents the activation of plasma damaging complement factors. This wasspeculated and assessed by comparing two different populations, Mediterranean and Northern ones,

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which showed that the healthier cardiometabolic features displayed by the first population may bedue to the higher concentration of plasma lutein as other antioxidants did not differ significantly [129].

Besides lutein and carotenoids, retinol-binding protein 4 (RBP4) might present beneficial effectwhen it is additionally supplemented. By measuring the circulating retinol-binding protein 4 (RBP4),transthyretin (TTR) and the cIMT, a link between vitamin A and subclinical atherosclerosis was shown.Further studies have proved that RBP4 links to insulin resistance, diabetes and atherosclerosis, retinolbeing inversely corelated with cIMT. Higher level of RBP4 was correlated with a specific role inatherogenesis, dietary vitamin A showing a strong protective action on subclinical atherosclerosis [130].

A study on clinically healthy European elder people hypothesized that vitamin A could belinked with atherosclerosis, endothelial function and left ventricular function, based on the newfindings that RBP4 concentration correlates with subclinical inflammation in the early stages of infantobesity. The study assessed cIMT and the plaque echogenity, revealing an inverse association betweencirculating RBP4 concentrations and cIMT which suggests the involvement of RBP4 in the atherogenicmechanism [131].

On the endothelial function, retinoids play an important role over nitric oxide, a potent vasodilatorthat controls vascular functions, but studies with human component are yet to take place.

A study on children aged 14–18 years showed elevated serum RBP4 concentration in obesepatients and strongly association with subclinical inflammation. Changes in lifestyle, such as physicalactivity and healthy diet, have decreased serum RBP4 concentration and the inflammatory markers,with important clinical implications [132].

The up-mentioned results are summarized in Table 4. Retinoid actions on atherosclerosis involvevarious cells and complex mechanisms, with multiple effects on the metabolic processes and responsesthat interact with atherogenesis and, implicitly, with subclinical atherosclerosis. The effects overcells and pathological situations reveal their specific biology, regulating both atherosclerotic andvascular injuries.

Table 4. Effects of vitamin A supplementation on subclinical atherosclerosis in patients withoutmanifest CVD.

Trial Year Population Age Results

Huang et al. [133] 2012709

postmenopausalwomen

52.9 ± 2.6 years No improvement oncIMT

Inglesson et al. [131] 2008 1008 healthysubjects 70 years Improvement on arterial

stiffness (cIMT)

2.6. Vitamin Supplementation Summary

This review aimed to assess the association between the vitamin intake and subclinicalatherosclerosis, trying to determine correlations between beneficial effects of rich-vitamin dietsand CVD development. Table 5 aims to offer a summary for the nowadays evidence and the possiblerecommendations for the vitamin supplementation effect on subclinical atherosclerosis in patientswithout manifest CVD as well as the major implications in atherogenesis for each vitamin. However, itis worth mentioning that the current CVD prevention guidelines do not recommend specific vitaminsupplementation. Nonetheless, being safe and effective to use, with low to none adverse effects, thesesubstances can be extremely valuable in the modern medicine focused on new alternative methods forthe prevention of atherothrombotic vascular disease.

Though the vitamin supplementation interventional trials presented divergent results, the lackin heterogeneity of study population, time of monitorization and different selected outcomes maybe key arguments for the up-to-date results. As well, there were more studies that tried to focus onincrease intake in secondary prevention than in primary prevention. Nonetheless, the interventionaltrials that regard subclinical atherosclerosis changes in patients without manifest CVD are rather

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few, but the results are promising and deserve further evaluation as subclinical disease often evolvesasymptomatic. Thus, for a better understanding and clear results of vitamin supplementation, futureclinical interventional trials should focus more on primary prevention in specific chosen grouppopulation. An individual comprehensive evaluation should be carefully assessed at baseline initiationfor having similar group characteristics, with the exclusion of several diseases that could alter thevitamin metabolism as renal or hepatic impairment. The follow-up should be conducted on longerperiods of time, at least 2–3 years, as the effects seem to be cumulative and time dependent. Further on,the dosage and type of administration should be standardized, and the final study analysis shouldaddress, as well, to different subgroups that may additionally benefit from the vitamin intake.

Table 5. Summary table for vitamin supplementation effect on subclinical atherosclerosis in subjectswithout CVD.

VitaminSupplementation

Level ofEvidence

Recommendation forSubclinical Atherosclerosis

ImprovementMain Implications in Atherogenesis

E +++ +++

↓ LDLc↓ oxidative stress↓ cellular

aggregability

↓ smooth muscle cellproliferation

↑atheroma plaquestabilization

D +++++ ++

↓ inflammation↓ oxidative stress↓ smooth musclecell proliferation

↓

renin-angiotensin-aldosteronesystem activity

C +++ +++↓ oxidized LDLc↓ oxidative stress↓ inflammation

↓monocyte endothelialadhesion↑ nitric oxide

↑ antioxidant effect

B complex + +↓ homocysteine↓ LDLc

↓ inflammation

↓ cellular aggregability↑ endothelial-dependent

vasodilatation↑ nitric oxide

A + +↓ oxidized LDLc↓ inflammation

↓ smooth muscle cellproliferation↑ nitric oxide

+ means the importance—where + is the least indicated/studied and +++++ is the most indicated/studied; ↓meansdiminish; ↑means augments.

3. Conclusions

Although certain clinical trials have shown promising results, there are so far no conclusive dataregarding the prevention and progression of atherosclerosis based on vitamin supplementation inpatients without CVD. However, given their enormous potential in this research area, future trialswill certainly bring additional information for a tailored CVD prevention focusing on early stages assubclinical atherosclerosis.

Author Contributions: Conceptualization—O.M., I.A.C., M.I., A.O.P., I.I.C.; investigation—A.I.L., I.M., I.A.C.; datacollection—D.C.D., A.D.C., I.A.C., O.M.; writing—original draft preparation—I.A.C., O.M., A.I.L., I.M., D.C.D.,A.D.C.; writing—review and editing—M.I., A.O.P., I.I.C.; visualization—O.M., M.I., D.C.D.; supervision—A.O.P.,I.I.C. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Conflicts of Interest: The authors declare no conflict of interest.

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